Composition for preventing Mycoplasma spp. infection

ABSTRACT

The present invention provides proteins that are suitable to be used as the active ingredient in subunit vaccine against Mycoplasma spp. The present invention also provides a subunit vaccine made therefrom. Said proteins have been experimentally proved to have the capability of inducing sufficient immune response to avoid pigs from Mycoplasma spp. infection. Said vaccine may have one of said proteins as active ingredient; or may have two or more of said proteins and is formulated as a cocktail vaccine. The present vaccine not only is safer than the conventional vaccines but also has equal or even better immune efficiency than the conventional ones. Furthermore, fusion partners suitable for producing said proteins of high solubility are also proved, which can significantly reduce production cost.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending U.S. application Ser. No.15/032,758, filed on Apr. 28, 2016, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/CN2013/087599, filedon Nov. 21, 2013, all of which are hereby expressly incorporated byreference into the present application.

BACKGROUND Technical Field

The present disclosure relates to a vaccine against Mycoplasma spp.;especially to a subunit vaccine against Mycoplasma spp.

Description of Related Art

Mycoplasma spp. is currently known the tiniest bacteria capable ofself-replication outside host cells. Although swine enzootic pneumoniawould not cause swine death, it will reduce feeding efficiency and causegrowth retardation, inflammation, and immunosuppression as well as makeswine more vulnerable to infection of other pathogens, which thereforebecome economic damage of the industry.

So far, swine enzootic pneumonia is prevented by three major strategies,including: medicine administration, environment management, andvaccination. Seeing the bad prevention efficiency of antibiotics toMycoplasma hyopneumoniae, medicine administration can only used fortreatment purposes and is hard to meet prevention needs. Furthermore,considering that drug abuse may lead to a larger infection causing bydrug-resistant bacteria, medicine administration needs cautious plansand exists a lot of limitations.

Environment management forms the basis of prevention of Mycoplasma spp.infection. Good piggery sanitation and management would be helpful toreduce occurrence of infection. On the other hand, prevention could bemore comprehensive through vaccination.

The conventional vaccines in the field use inactive/dead bacteria as theactive ingredient thereof. However, the price of the conventionalvaccines is too high because Mycoplasma spp. is fastidious bacteria andis to difficult to be cultured in the laboratory. In order to reduce thecost of Mycoplasma spp. vaccines, scientists continuously try to developvaccines of different types, such as: (1) attenuated vaccines, (2)vector vaccines, (3) subunit vaccines, and (4) DNA vaccines. Among them,subunit vaccines show the most potential because the advantages of easein production and high safety.

To date, there are several potential candidate proteins that could beused for M. hyopneumoniae vaccines; however, there is no further reportverifying the proteins suitable for M. hyopneumoniae vaccines.

SUMMARY

In light of the foregoing, one of the objects of the present inventionis to provide antigens suitable for being used in Mycoplasma spp.vaccines and thereby producing novel Mycoplasma spp. vaccines so thatthe cost of prevention can be reduced.

Another object of the present invention is to provide a combination ofantigens that suitable for being used in Mycoplasma spp. vaccines andthereby provide subunit vaccines with better performance; therefore,there would be more options for prevention tasks.

In order to achieve the aforesaid objects, the present inventionprovides a protein for preventing Mycoplasma spp. infection, comprisingan amino acid sequence of SEQ ID NO: 01, SEQ ID NO: 02, or a combinationthereof.

The present invention also provides a composition for preventingMycoplasma spp. infection, comprising: a first active ingredient,comprising a protein of P46, Tuf, or a combination thereof; and apharmaceutically acceptable adjuvant.

Preferably, said first active ingredient has an amino acid sequence ofSEQ ID NO: 01, SEQ ID NO: 02, or a combination thereof.

Preferably, said composition further comprises a second activeingredient, comprising a protein of MHP30, NrdFC, or a combinationthereof.

Preferably, said second active ingredient has an amino acid sequence ofSEQ ID NO: 03, SEQ ID NO: 04, or a combination thereof.

Preferably, said first active ingredient and/or said second activeingredient is independently of a concentration of 20 to 2000 μg/mL basedon the total volume of said composition.

Preferably, said pharmaceutically acceptable adjuvant is a completeFreund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, asurfactant, a polyanion adjuvant, a peptide, an oil emulsion, or acombination thereof.

Preferably, said composition further comprises a pharmaceuticallyacceptable additive.

Preferably, said pharmaceutically acceptable additive is a solvent, astabilizer, a diluent, a preservative, an antibacterial agent, anantifungal agent, an isotonic agent, an absorption delaying agent, or acombination thereof.

The present invention further provides a composition for preventingMycoplasma spp. infection, comprising: an active ingredient, having atleast two proteins selected from a group consisting of P46, Tuf, MHP30,and NrdFC; and a pharmaceutically acceptable adjuvant.

Preferably, said active ingredient is P46, Tuf, MHP30, and NrdFC.

Preferably, said active ingredient has at least two amino acid sequencesselected form a group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ IDNO: 03, and SEQ ID NO: 04.

Preferably, said active ingredient has amino acid sequences of SEQ IDNO: 01, SEQ ID NO: 02, SEQ ID NO: 03, and SEQ ID NO: 04.

Preferably, said active ingredient is of a concentration of 20 to 2000μg/mL based on the total volume of said composition.

Preferably, said pharmaceutically acceptable adjuvant is a completeFreund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, asurfactant, a polyanion adjuvant, a peptide, an oil emulsion, or acombination thereof.

Preferably, said composition further comprises a pharmaceuticallyacceptable additive.

Preferably, said pharmaceutically acceptable additive is a solvent, astabilizer, a diluent, a preservative, an antibacterial agent, anantifungal agent, an isotonic agent, an absorption delaying agent, or acombination thereof.

The present invention also provides an expression vector for preparingthe aforesaid active ingredient; wherein said expression vectorcomprises a plasmid; wherein said plasmid comprises: a nucleotidesequence comprising at least one sequence selected from a groupconsisting of SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, and SEQ IDNO: 08; a gene encoding a fusion partner, selected from a groupconsisting of MsyB of E. coli, YjgD of E. coli, GroS17 of E. coli, GroESof Bacillus subtilis, TrxA of Alicyclobacillus acidocaldarius, SUMO ofS. cerevisiae, and Vgb of Vitreoscilla spp.; and a regulatory element.

Preferably, said regulatory element comprises a promoter and a ribosomebinding site.

Preferably, said plasmid is pET-MSY, pET-YjgD, pET-GroS17, pET-GroES,pET-TrxA, pET-SUMO, or pET-Vgb.

Preferably, regarding said expression vector, provided that, when saidnucleotide sequence is at least one sequence selected from a groupconsisting of SEQ ID NO: 05, SEQ ID NO: 06, and SEQ ID NO: 08, said geneencoding a fusion partner is MsyB of E. coli.

Preferably, regarding said expression vector, provided that, when saidnucleotide sequence is SEQ ID NO: 07, said gene encoding a fusionpartner is selected from a group consisting of YjgD of E. coli, GroS17of E. coli, GroES of Bacillus subtilis, TrxA of Alicyclobacillusacidocaldarius, SUMO of S. cerevisiae, and Vgb of Vitreoscilla spp.

Preferably, said expression vector is applied for used for an E. coligene expression system.

The present invention further provides a method for preparing solubleantigen, comprising using the aforesaid expression vector; wherein saidantigen is P46, Tuf, MHP30, or NrdFC.

To sum up, the present invention is related to antigens, composition,and expression vector for preparing said antigen for preventing fromMycoplasma spp. infection. The present disclosure not only provides newoptions for prevention tasks, but also proves that a “cocktail” subunitvaccine of combining at least two antigens (that is, having at least twoantigens as active ingredients) is able to enhance the immune induction.Moreover, the present invention teaches some fusion partners that areparticularly suitable for assisting the expression of the aforesaidantigens in an expression system so that are favorable for increasingthe production capacity of preparing the required vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a result of a protein electrophoresis, showing various fusionpartners have different effects on the solubility of MHP 30 prepared.Line M: protein marker (PageRuler™ pretained protein ladder; Fermentas,USA); Line 1: E. coli BL21 (DE3) (pET-YjgD-MHP30), soluble protein; Line2: E. coli BL21 (DE3) (pET-SUMO-MHP30), soluble protein; Line 3: E. coliBL21 (DE3) (pET-GroS17-MHP30), soluble protein; Line 4: E. coli BL21(DE3) (pET-GroES-MHP30), soluble protein; Line 5: E. coli BL21 (DE3)(pET-D-MHP30), soluble protein; Line 6: E. coli BL21 (DE3)(pET-MsyB-MHP30), soluble protein; Line 7: E. coli BL21 (DE3)(pET-Vgb-MHP30), soluble protein; Line 8: E. coli BL21 (DE3)(pET-TrxA-MHP30), soluble protein.

FIG. 2 is a result of a protein electrophoresis, showing various fusionpartners have different effects on the solubility of P46, NrdFC, and Tufprepared. Line M: protein marker (PageRuler™ pretained protein ladder;Fermentas, USA); Line 1: E. coli BL21 (DE3) (pET-D-P46M), solubleprotein; Line 2: E. coli BL21 (DE3) (pET-D-P46M), insoluble protein;Line 3: E. coli BL21 (DE3) (pET-D-NrdFC), soluble protein; Line 4: E.coli BL21 (DE3) (pET-D-NrdFC), insoluble protein; Line 5: E. coli BL21(DE3) (pET-D-Tuf), soluble protein; Line 6: E. coli BL21 (DE3)(pET-D-Tuf), insoluble protein; Line 7: E. coli BL21 (DE3)(pET-MysB-P46M), soluble protein; Line 8: E. coli BL21 (DE3)(pET-MysB-P46M), insoluble protein; Line 9: E. coli BL21 (DE3)(pET-MysB-NrdFC), soluble protein; Line 10: E. coli BL21 (DE3)(pET-MysB-NrdFC), insoluble protein; Line 11: E. coli BL21 (DE3)(pET-MysB-Tuf), soluble protein; Line 12: E. coli BL21 (DE3)(pET-MysB-Tuf), insoluble protein.

FIG. 3 is a result of a protein electrophoresis, showing thepurification of the present recombinant proteins MHP30, P46, NrdFC, andTuf. Line M: protein marker (PageRuler™ pretained protein ladder;Fermentas, USA); Line 1: E. coli BL21 (DE3) (pET-YjgD-MHP30), solubleprotein; Line 2: E. coli BL21 (DE3) (pET-MysB-P46M), soluble protein;Line 3: E. coli BL21 (DE3) (pET-MysB-NrdFC), soluble protein; Line 4: E.coli BL21 (DE3) (pET-MysB-Tuf), soluble protein; Line 5: purified MHP30fusion protein; Line 6: purified P46 fusion protein; Line 7: purifiedNrdFC fusion protein; Line 8: purified Tuf fusion protein.

FIG. 4 shows the result of Western Blot assay, showing that miceantiserum identified the total cell lysate of M. hyopneumoniae.

FIG. 5 shows the result of ELISA assay, showing the results of theimmune challenge experiments conducted in Embodiment 7 of the presentinvention.

DETAILED DESCRIPTION

The present invention is related to antigens, composition, andexpression vector for preparing said antigen for preventing fromMycoplasma spp. infection. Specifically, the present invention provesthe antigenic effect of P46 and Tuf for preparing composition forMycoplasma spp. infection prevention. Furthermore, the present inventionalso proves a “cocktail” subunit vaccine of combining at least two ofP46, Tuf, MHP30, and NrdFC as active ingredients is able to enhance theimmune induction. On the other hand, the present invention disclosesfusion partners, which are particularly suitable for preparing P46, Tuf,MHP30, and NrdFC of high solubility (ie. water-solubility). Throughusing the aforesaid fusion partners, the time and cost required forpreparing vaccines can be significantly reduced.

In one aspect of the present invention, the present invention providescomposition for preventing Mycoplasma spp. infection, comprising: afirst active ingredient, comprising a protein of P46, Tuf, or acombination thereof; and a pharmaceutically acceptable adjuvant. In analternative embodiment, said composition may further comprise a secondactive ingredient. Said second active ingredient comprises a protein ofMHP30, NrdFC, or a combination thereof.

In another aspect of the present invention, the present inventionprovides a composition for preventing Mycoplasma spp. infection,comprising: an active ingredient, having at least two proteins selectedfrom a group consisting of P46, Tuf, MHP30, and NrdFC; and apharmaceutically acceptable adjuvant.

In an alternative embodiment of the present invention, said P46 iscorresponding to the amino acid sequence showed as SEQ ID NO: 01; saidTuf is corresponding to the amino acid sequence showed as SEQ ID NO: 02;said MHP30 is corresponding to the amino acid sequence showed as SEQ IDNO: 03; said NrdFC is corresponding to the amino acid sequence showed asSEQ ID NO: 04. Those having ordinary skill in the art can readilyunderstand that as long as the antigenic determinant formed by foldingof a peptide of said amino acid sequence is not interfered, said activeingredient may be a fusion protein with at least two said sequences.

Typically, combining two or more antigens in one single vaccine is notalways favorable for the immune induction of the vaccine. In fact,combining two or more antigens in one single vaccine may cause undesiredsituation that the immune induction of the two or more antigens conflictwith each other and are thereby reduced. Besides, from the perspectiveof cost, even if not conflicting with each other, if the immuneinduction of the two or more antigens do not exhibit synergistic effect,it would be worthless to combine two or more antigens in one singlevaccine. The researches of the present invention proved that P46, Tuf,MHP30, and NrdFC exhibited better immune induction while be used incombination. Therefore, in an alternative embodiment, said activeingredient of the present composition comprises any two or more of theaforesaid proteins; that is, the cocktail vaccine of the presentinvention.

The concentration of said active ingredient(s) in the presentcomposition is 20 to 2000 μg/mL based on the total volume of saidcomposition. In a preferable embodiment of the present invention, oneactive ingredient in the present composition is of a concentration of 20to 500 μg/mL based on the total volume of said composition. In analternative embodiment of the present invention, the present compositioncontains at least one of said proteins as active ingredients; whereinthe total concentration of those active ingredients is 20 to 1000 μg/mL,20 to 1500 μg/mL or 20 to 2000 μg/mL based on the total volume of saidcomposition.

Said pharmaceutically acceptable adjuvant is used for improving theimmune effect of said active ingredient, stabilizing said activeingredient, and/or increasing the safety of vaccines. Saidpharmaceutically acceptable adjuvant of the present invention includes,but not limits to: a complete Freund's adjuvant, an incomplete Freund'sadjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide,an oil emulsion, or a combination thereof.

Another aspect of the present invention is related to an expressionvector. Specifically, said expression vector is used for an E. coliexpression system. In other words, said expression vector is able to betranslated into a peptide of amino acid sequence of desired protein, andthe peptide can fold forming the desired active ingredient required forthe present composition. However, based on the spirit of the presentinvention, those having ordinary skill in the art can refer to thedisclosure of the present invention and make modification accordingly tofit in various expression system while still belong to the scope of thepresent invention (for instance, making modification to the sequences inorder to correspond to different codon usage).

Another aspect of the present invention is related to a method forpreparing a protein for preventing from Mycoplasma spp. infection byusing said expression vector. Proteins prepared by the conventionalexpression vectors in the field usually have drawback of insolubility;therefore it is necessary to treat and solve the purified product byurea and guanidinium hydrochloride. However, those treatments not onlyincrease production cost but also cause denature of the proteins so thatre-folding is sometimes needed for those denatured proteins for regaintheir antigenic function. While there is certain possibility that there-folding process may fail, the antigenic effect of those proteinsmight be reduced. In light of the insufficiency of the aforesaidconventional preparation, the present method has strength in matchingfusion partner that is particularly suitable for providing excellentsolubility to the aforesaid proteins for Mycoplasma spp. infectionprevention. Therefore, the production cost can be saved, the preparationprocedures can be simplified, and the efficacy of the produced proteinsof being the active ingredients of vaccines can be enhanced.

In an alternative embodiment of the present invention, said expressionvector comprises a plasmid. Said plasmid comprises: a nucleotidesequence, a gene encoding a fusion partner and a regulatory element.Said nucleotide sequence comprises at least one sequence selected from agroup consisting of SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, and SEQID NO: 08, which respectively correspond to P46, Tuf, MHP30, and NrdFC.

In an alternative embodiment, as long as the operation of the geneexpression system is not hindered and the production of said nucleotidesequence and the folding of the consequent amino acid sequence thereofare not interfered, said plasmid may comprise two or more saidnucleotide sequences.

Said gene encoding a fusion partner is selected from a group consistingof MsyB of E. coli, YjgD of E. coli, GroS17 of E. coli, GroES ofBacillus subtilis, TrxA of Alicyclobacillus acidocaldarius, SUMO of S.cerevisiae, and Vgb of Vitreoscilla spp.

Said regulatory element is referred to an element required forinitiating the transcription and translation in the expression system.Said regulatory element shall at least comprise a promoter, and aribosome binding site. Preferably, said regulatory element may furthercomprise: an operator, an enhancer sequence, or a combination thereof.

The following examples recite the trials and experiments of the presentinvention in order to further explain the features and advantages of thepresent invention. It shall be noted that the following examples areexemplary and shall not be used for limiting the claim scope of thepresent invention.

Example 1: Strains and Culture Thereof

Current researches identified there are seven kinds of Mycoplasm spp.isolated from swines: Mycoplasm hyopneumoniae, Mycoplasma hyorhinis,Mycoplasma hyosynoviae, Mycoplasma flocculare, Mycoplasma hyopharyngis,Mycoplasma sualvi, and Mycoplasma bovigenitalium (Gourlay et al., 1978;Blank et al., 1996; Assuncao et al., 2005). Among them, Mycoplasmhyopneumoniae is the main pathogen causing swine 25 mycoplasmapneumonia. The infection rate is somewhere in between 25% to 93%.Therefore, the present research used Mycoplasm hyopneumoniae (Mycoplasmhyopneumoniae, PRIT-5) as the source of antigen gene. In addition, thepresent research used Escherichia coli JM109 as the host for genecloning and Escherichia coli BL21 (DE3) as host for protein expression.

M. hyopneumoniae was cultured by using Friis medium (Friis et al.,1975). Escherichia coli (E. coli) strains were cultured by using LBmedium (Luria-Bertani). Proper amount of antibiotic and/or 1.5% of agarfor preparing solid medium were optionally added in accordance theexperiment conditions.

Example 2: Cloning of M. hyopneumoniae Antigen Gene

Specific primers were designed for different antigen genes (Table 1).The genome of M. hyopneumoniae was used as template and amplificationwas conducted by using the aforesaid primers. The details of geneamplification and cloning were described in the following paragraphs:

TABLE 1 Primer sets for amplifying the desired genes. Desired genesSequence of primer (5′ to 3′) MHP30 MHP30F (SEQ ID NO 09):GATATAGGATCCGCAAAATTAGACGATAATCTTCAGTATTCA MHP30R (SEQ ID NO 10):CAATATGTCGACTTAATTTTTACCTTGTTTTTTAATGATTTCGTC P46 P46F (SEQ ID NO 11):GATATAGGATCCATGAAAAAAATGCTTAGAAAAAAATTCTTG P46R (SEQ ID NO 12):CAATATGTCGACTTAGGCATCAGGATTATCAACATTAGC NrdFC NrdCF (SEQ ID NO 13):GATATAGGATCCGATCTATTATATAAACTAATTGAATTAGAAAAAGATTATCNrdCR(SEQ ID NO 14): CAATATGTCGACTTAAAACTCCCAATCTTCATCTTCG TufTufF (SEQ ID NO 15): GATATAGGATCCATGGCAGTTGTTAAAACGACAGGAAAATufR (SEQ ID NO 16): CAATATGTCGACTTATTTAATAATTTCGGTAACTGTTCCGGCA*GGATCC: BamHI cutting site; GTCGAC: SalI cutting site1. Extraction of M. hyopneumoniae Genome.

The extraction of M. hyopneumoniae genome was conducted by using DNApurification kit (Tissue & Cell Genomic DNA Purification kit; GeneMark,Taiwan). First, 4.5 mL of broth was put under centrifugation (5,870×g, 5min) in tubes to discard the supernatant and collect the pellet. Then,20 μL of proteinase K (10 mg/mL) and 200 μL of extraction reagent wereadded in for reacting at 56° C. for 3 hours. The pellet and theaforesaid reagents were mixed by up-side-down or shaking to the tubeevery 5 minutes during the period to make sure they were well-mixed.

After reaction with the extraction reagent, the solution becametransparent as the pellet was digested completely. 200 μL of bindingreagent was then added in for reacting at 70° C. for 10 minutes. Afterthat, 200 μL of absolute ethanol was added into the tube and mixed. Allthe contents inside the tube were moved to a spin column and the spincolumn was then positioned in a collection tube. After centrifugation(17,970×g) for 2 minutes, the effluent was discarded and 700 μL of washsolution was added into the spin column. After another centrifugation(17,970×g) for 2 minutes, the effluent was discarded and the aforesaidprocedure was repeated again. Lastly, the tube was put undercentrifugation (17,970×g) for 5 minutes to remove residual ethanol.Then, the spin column was position in a sterile tube and a proper amountof sterile water was added in for elute the DNA.

The concentration of the purified M. hyopneumoniae genomic DNA wasdetermined by using Quant-iT™ dsDNA High-Sensitivity Assay Kit(Invitrogen, Madison, USA) and Qubit Fluorometer (Invitrogen, Madison,USA). The operation is: mixing Quant-iT reagent and Quant-iT buffer at aratio of 1:200 to obtain a working solution. 190 μL of working solutionand 10 μL of the standard sample were mixed and placed in roomtemperature for 2 minutes. Then, a standard curve was depicted. Afterthat, 2 μL of sample and 198 μL of working solution were mixed, placedfor 2 minutes and detected by Qubit Fluorometer for determining theconcentration of the genomic DNA. The calculation formula of theconcentration (ng/μL) was: measured value×100.

2. Amplification of Desired Genes by Polymerase Chain Reaction (PCR)

The M. hyopneumoniae genomic DNA was used as template and primersdesigned for P46 gene, Tuf gene, MHP30 gene, and NrdFC gene wererespectively used for PCR reaction. Each primer used and conditions forthe PCR reaction were shown in the following Table 2 (the sequences ofthe primers were listed in Table 1 above). 50 μL of PCR mixturecontained 1×GDP-HiFi PCR buffer B, 200 μM of mixture of dATP, dTTP,dGTP, and dCTP, 1 μM amplification primer, 200 ng of M. hyopneumoniaegenomic DNA and 1 U of GDP-HiFi DNA polymerase. After the PCR reaction,gel electrophoresis was conducted to confirm the existing of theamplified DNA fragment of expected size.

TABLE 2 PCR conditions and primer sets Desired genes Primer setConditions MHP30 MHP30F 98° C. for 5 minutes (one cycle); 94° C. for 30seconds, 55° C. for 30 MHP30R seconds, 68° C. for 30 seconds (35cycles); 68° C. for 5 minutes (one cycle). P46 P46F 98° C. for 5 minutes(one cycle); 94° C. for 30 seconds, 55° C. for 30 P46R seconds, 68° C.for 45 seconds (35 cycles); 68° C. for 5 minutes (one cycle). NrdFCNrdCF 98° C. for 5 minutes (one cycle); 94° C. for 30 seconds, 55° C.for 30 NrdCR seconds, 68° C. for 30 seconds (35 cycles); 68° C. for 5minutes (one cycle). Tuf TufF 98° C. for 5 minutes (one cycle); 94° C.for 30 seconds, 55° C. for 30 TufR seconds, 68° C. for 45 seconds (35cycles); 68° C. for 5 minutes (one cycle).3. Collection of the PCR Product and Cloning Thereof.

The collection of the PCR products was made by using the PCR-M™ Clean Upsystem kit (GeneMark, Taiwan; the experiments were conducted byfollowing the operation manual and were not reiterated here). Then, thecloning was made by using the CloneJET PCR Cloning Kit. The experimentsof cloning were conducted according to the operation manual, which werebriefly described as follows. Firstly, the collected PCR product wasmixed with the reagents and DNA ligase of the kit for conductingligation reaction at 22° C. for 30 minutes. The ligation mixture wasthen transformed into E. coli strain ECOS™ 9-5 (Yeastern, Taiwan). Theconditions of transformation were referred to the manual. The bacteriatransformed were transferred into 1 mL of SOC recovering medium andcultured at 37° C. with shaking at 250 rpm. A proper amount of broth wasplating on LB solid medium plates with ampicillin (100 μg/mL) and theplates were cultured at 37° C. for 16 hours. After that, a colony PCRwas conducted to screen the strains, whose transformation succeeded. Thecolony PCR was conducted as following steps. First of all, a tubecontaining PCR mixture of 50 μL of 2× Taq PCR MasterMix (Genomics,Taiwan), 0.5 μL of forward primer for amplifying the desired gene, 0.5μL of reverse primer for amplifying the desired gene, and 49 μL sterilewater was prepared. After mixing, the PCR mixture was distributed toseveral PCR tubes (10 μL/tube). Colonies on the aforesaid plates wererandomly picked into the PCR tubes respectively for PCR reaction. Theconditions of PCR reaction are: 95° C. for 5 minutes (one cycle); 95° C.for 30 seconds, 55° C. for 30 seconds, 72° C. for X minutes (25 cycles);72° C. for 7 minutes (one cycle); wherein the “X” was depended on theextension time required for the DNA polymerase and was set according tothe size of the fragment to be amplified. The extension rate of Taq DNApolymerase is 1 kb/min; therefore, if the fragment to be amplified has asize of about 1 kb, the “X” would be set as 1 minute. After reaction, agel electrophoresis was conducted to confirm the PCR results. For thosetransformation strains being confirmed successfully had insert DNA intheir recombinant plasmids, the plasmids were extracted out for DNAsequencing. The plasmids have MHP30 gene, P46 gene, NrdFC gene, and Tufgene were respectively named as pJET-MHP30, pJET-P46, pJET-NrdFC, andpJET-Tuf.

The DNA sequencing results showed that the P46 coloned in the presentinvention has an amino sequence and nucleotide sequence as SEQ ID NO 01and SEQ ID NO 05 respectively. Tuf has an amino sequence and nucleotidesequence as SEQ ID NO 02 and SEQ ID NO 06 respectively. MHP30 has anamino sequence and nucleotide sequence as SEQ ID NO 03 and SEQ ID NO 07respectively. NrdFC has an amino sequence and nucleotide sequence as SEQID NO 04 and SEQ ID NO 08, respectively.

Example 3: Point Mutation and Cloning of P46 Gene

P46 has three TGA codons. TGA codon was translated as tryptophan inMycoplasma spp. but as stop codon in E. coli. In order to prevent fromfailure in using E. coli expression system for producing the wholedesired protein, point mutation must be made to P46 gene's TGA codon toreplace them as TGG, which is translated by E. coli as tryptophan.

The principle for designing mutation primers for the point mutation isthe mutation point shall be located in the centre area of the primer andthe Tm of the primers shall be higher than 78° C. The Tm of the primerscan be calculated by the formula provided by Invitrogene: Tm=81.5+0.41(% GC)−675/N−% mismatch. “% GC” stands for the percentage of G and C ofthe primer concerned; “N” stands for the length of the primer concerned;“% mismatch” stands for the percentage of the base to be mutated of theprimer concerned. The primers used for point mutation of P46 gene werelisted in the following Table 3, including P46F/P46M2, P46M1/P46M4,P46M3/P46M6 and P46M5/P46R.

TABLE 3 Primers used for point mutation of P46 gene Desired genesSequence of primer (5′ to 3′) P46F SEQ ID NO 11:GATATAGGATCCATGAAAAAAATGCTTAGAAAAAAATTCTTG P46M2 SEQ ID NO 17:CTCTTTGGGCACTAATCCATCGAGGATTATCCGG P46M1 SEQ ID NO 18:CCGGATAATCCTCGATGGATTAGTGCCCAAAGAG P46M4 SEQ ID NO 19:ATTAGCTTGCTGAGTGAGCCAGTTATTTTGTGCATCC P46M3 SEQ ID NO 20:GGATGCACAAAATAACTGGCTCACTCAGCAAGCTAAT P46M6 SEQ ID NO 21:CGGCAGTTCCATAATTCCATCCTGGGACATAAAC P46M5 SEQ ID NO 22:GTTTATGTCCCAGGATGGAATTATGGAACTGCCG P46R SEQ ID NO 12:CAATATGTCGACTTAGGCATCAGGATTATCAACATTAGC

The 50 μL of PCR mixture contained 1×GDP-HiFi PCR buffer B, 200 μM ofmixture of dATP, dTTP, dGTP, and dCTP, 1 μM of amplification primer, 100ng of pJET-P46 and 1 U GDP-HiFi DNA polymerase. The PCR reactionconditions were: 98° C. for 2 minutes (one cycle); 94° C. for 30seconds, 55° C. for 30 seconds, 68° C. for 45 seconds (35 cycles); 68°C. for 5 minutes (one cycle). After the PCR reaction, gelelectrophoresis was conducted to confirm the existing of the amplifiedDNA fragment of expected size.

The PCR products were collected by using Gel-M™ gel extraction systemkit and the experiments were conducted according to the manual. Then,the four PCR products collected were used as the templates for geneamplification with P46F/P46R primer set. The PCR reaction conditionswere: 98° C. for 2 minutes (one cycle); 94° C. for 30 seconds, 55° C.for 30 seconds, 68° C. for 45 seconds (35 cycles); 68° C. for 5 minutes(one cycle). After that, the whole length of the P46 gene being pointmutated can be obtained. The PCR products were collected by using PCR-M™Clean Up System Kit (GeneMark, Taiwan). And the cloning of the mutationgene was made by using CloneJET PCR Cloning Kit. For thosetransformation strains being confirmed, by colony PCR, successfully hadinsert DNA in their recombinant plasmids, the plasmids were extractedfor DNA sequencing. The plasmid having the mutated P46 gene was named aspJET-P46M.

Example 4: Establishment of the Present M. hyopneumoniae AntigenExpression Vector

Plasmids having various fusion partners were used as backbone forestablishing M. hyopneumoniae antigen expression vector. The fusionpartner genes used were MsyB of E. coli, YjgD of E. coli, the partialpeptide of GroS containing 17 amino acids (GroS17) of E. coli, GroES ofBacillus subtilis, TrxA of Alicyclobacillus acidocaldarius, SUMO of S.cerevisiae, D protein of bacteriophage phiX174, and Vgb of Vitreoscillaspp. The establishment was made as follows:

1. The Establishment of MHP30 Expression Vector.

Cutting pJET-MHP30 with BamHI and SalI and the resulted DNA fragment wasligased into a fusion expression plasmid pre-cut by the same restrictionenzymes. The ligation product was than transformed into E. coli ECOS9-5. Transformation strains were screened by colony PCR and confirmingif there was DNA fragments of expected size by using DNAelectrophoresis. For those transformation strains being confirmedsuccessfully had insert DNA in their recombinant plasmids, the plasmidswere extracted out for DNA sequencing. The plasmids having correct DNAsequence were respectively named as pET-MSY-MHP30, pET-YjgD-MHP30,pET-GroS17-MHP30, pET-GroES-MHP30, pET-TrxA-MHP30, pET-SUMO-MHP30,pET-D-MHP30, and pET-Vgb-MHP30.

2. The Establishment of P46 Expression Vector.

Cutting pJET-P46M with BamHI and SalI and the following experiments wereconducted by referring to the aforesaid establishment steps of MHP30expression vector. The plasmids having correct DNA sequence wererespectively named as pET-D-P46M, and pET-MSY-P46M.

3. The Establishment of NrdFC Expression Vector.

Cutting pJET-NrdFC with BamHI and SalI and the following experimentswere conducted by referring to the aforesaid establishment steps ofMHP30 expression vector. The plasmids having correct DNA sequence wererespectively named as pET-D-NrdFC, and pET-MSY-NrdFC.

4. The Establishment of Tuf Expression Vector.

Cutting pJET-Tuf with BamHI and SalI and the following experiments wereconducted by referring to the aforesaid establishment steps of MHP30expression vector. The plasmids having correct DNA sequence wererespectively named as pET-D-Tuf, and pET-MSY-Tuf.

Example 5: Expression of Recombinant M. hyopneumoniae Antigens andPurification Thereof

Regarding to Expression of the Desired Antigens

The antigen expression vectors were transformed into E. coli BL21 (DE3).One single colony was picked and inoculate on LB medium containingkanamycin of final concentration of 30 μg/mL and cultured underconditions of 37° C. and 180 rpm overnight. Then the broth wasinoculated into fresh LB medium (containing kanamycin of finalconcentration of 30 μg/mL) at a ratio of 1:100 and cultured conditionsof 37° C. and 180 rpm until the OD₆₀₀ value thereof achieving around0.6˜0.8. 0.1 mM of IPTG was added in for inducing the expression of thedesired protein for 4 hours. Then, the broth was put undercentrifugation (10,000×g, 10 minutes, 4° C.) and the pellet wascollected. After that, the soluble portion and the insoluble portionthereof were separated by using Easy-Lyse Bacterial Protein Extractionkit (Epicentre, USA). Protein electrophoresis was then conducted toobserve the solubility of the produced recombinant antigens.

FIGS. 1 and 2 respectively showed different solubility of MHP30 uponusing various fusion partners in expression; and different solubility ofP46, NrdFC, and Tuf upon using various fusion partners in expression.The results indicated that using YjgD, SUMO, GroS17, GroES, Vgb, or TrxAas fusion partner can increase the solubility of MHP30. On the otherhand, using MsyB as fusion partner can provide P46, NrdFC, and Tufexcellent solubility.

Regarding to Purification of the Desired Antigens

Taking the advantage of the fact that the N′ Hig tag of recombinantprotein can form coordinate covalent bond with nickel or cobalt ion,immobilized-metal affinity chromatography (IMAC) was used for proteinpurification. The protocol of protein purification was referred to TheQIA Expressionist™ (fourth edition, Qiagen). The pellet was suspended inlysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) andbroken by homogenizer. Then, centrifugation was conducted and thesupernatant was collected. The collected supernatant was introduced intoa resin column (1 mL Ni-NTA) where the recombinant antigen would attachon. After that, 15 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mMimidazole, pH 8.0) was added in to wash the resin inside the column toremove non-specific binding of proteins. Lastly, 20 mL of elution buffer(50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0) was introduced toelute the antigens on the resin. The elution buffer contained highconcentration of imidazole to compete the binding site on the resin withthe recombinant proteins so that the recombinant proteins can be washedoff from the resin. The result of purification was observed by proteinelectrophoresis. According to FIG. 3, it clearly showed that the His tagtagged on N′ of the present recombinant antigens was indeed favorablefor purifying the present recombinant antigens by IMAC.

Example 6: Mice Antiserum Trials

This example used 5-week BALB/c female mice (National Laboratory AnimalCenter). The mice were maintained in standard animal room with airconditioner and offered with food and clean water. The immune challengewas conducted after the mice being raised 7 days. The recombinantantigens purified in the aforesaid examples was mixed with adjuvant as amixture (complete Freund's adjuvant or incomplete Freund's adjuvant)through a three-way connector and subcutaneously injected into the mice.The first injection was a complete Freund's adjuvant formulation. After7 days from the first injection, the mice were injected with incompleteFreund's adjuvant formulation twice in a 7 days interval. After 7 daysfrom the second injection, blood was collected from eye orbit of themice.

The collected blood samples were placed in room temperature for 1 hourand placed at 4° C. overnight to let the blood coagulate. Then, thesamples were put under centrifugation (5,000×g) for 30 minute to obtainthe antiserum, which contained specific antibody against the recombinantantigen.

The obtained antiserum was then used to against total cell lysate of M.hyopneumoniae. The experiment protocol was: suspending M. hyopneumoniaePRIT-5 in SET buffer (50 mM glucose, 25 mM-HCl, 10 mM EDTA, pH 8.0) andadding 5× sample buffer (312.5 mM Tris-HCl (pH 6.8), 50% glycerol, 10%SDS, 0.05% bromophenol blue); heating the sample at 100° C. for 5minutes after the sample was well-mixed with the aforesaid buffer. Then,protein electrophoresis was conducted. The gel, after proteinelectrophoresis, was immersed in transfer buffer (25 mM Tris base, 192mM glycine, 10% (v/v) methanol, pH 8.3). PVDF membrane of suitable sizewas rinsed with methanol for few seconds, washed by deionized water, andthen immersed into transfer buffer. After the gel and PVDF membrane wereboth immersed in transfer buffer for 15 minutes, a filter paper, thegel, the PVDF membrane and another filter paper were sequentially placedin a Trans-Blot SD Semi-Dry Transfer Cell for transferring undersuitable electric supply. After the transferring was done, the PVDFmembrane was immersed in 20 mL of block buffer (20 mM Tris, 150 mM NaCl,5% skim milk, pH7.4) in room temperature for 1 hour and 10 μL ofrecombinant antigen was added in (2000× diluation). After shaking inroom temperature for 1 hour, the block buffer was discarded and the PVDFmembrane was by 20 mL TBST buffer (20 mM Tris, 150 mM NaCl, 0.05%Tween-20, pH7.4) three times (5 minute for each time). 20 mL of blockbuffer and 4 μL of alkaline phosphatase-conjugated goat anti-mouse IgG(H+L) were then added in and the mixture was shaken in room temperaturefor 1 hour. After that, the PVDF membrane was washed by TBST bufferthree times and NBT/BCIP reagent was added in for detection.

The result showed that the mixture of the present recombinant antigenand suitable adjuvant did induce antibody against the antigen in mice.That is to said, the present proteins had potential to be immunogens(FIG. 4).

Example 7: Swine Immune Tests and Challenge Tests

1. Vaccine Preparation.

One or more of the purified recombinant antigens of the aforesaidexamples were mixed with an adjuvant to obtain desired subunit vaccinesor cocktail vaccines. The dosage of every vaccine prepared was 2 mL,which contained 200 μg of each kind of the present proteins added.

2. Swine Immune Tests.

The aforesaid subunit vaccines or cocktail vaccines were used for swineimmune tests. 2-weeks SPF swine were bought from the second generationSPF swine house of Animal Technology Institute Taiwan. All the swinewere maintained and fed equally in the SPF swine house.

The vaccines (2 mL) were intramuscularly injected to the swine when theyreached 21-day age and blood thereof was collected at 21, 42, 70, and98-day age for isolating serum. Mycoplasma spp. antibody trials (IDEXX)kit was used for analyzing the collected serum antibodies. The operationwas as the follows. First of all, 10 μL of serum was mixed with 390 μLof sample diluents gently to obtain a mixture. 100 μL of the mixture wasadded in the wells of a 96-well plate, which already had Mycoplasma spp.antigen inside. Besides, 100 μL of positive serum and negative serumwere also added to different wells of the plate as controls. The platewas placed in room temperature for 30 minutes. Then, the liquid in theplate was discarded and 350 μL of wash buffer was added in three timesfor washing. 350 μL of horseradish peroxidase labeled with anti-swineantibodies was then added in to the plate and the plate was placed inroom temperature for 30 minutes. After that, the liquid in the plate wasdiscarded and 350 μL of wash buffer was added in three times forwashing. 100 μL of TMB substrate solution was added into the plate andthe plate was placed in room temperature for 15 minutes. Then, 100 μL ofstop buffer was added to terminate the color reaction. Lastly, theabsorbance of each well was evaluated at 650 nm by an ELISA reader forcalculation of S/P value. S/P value=(OD₆₅₀ absorbance of trialgroup−OD₆₅₀ absorbance of negative control group)/(OD₆₅₀ absorbance ofpositive control group−OD₆₅₀ absorbance of negative control group). Thehigher the S/P value, the more the antibodies against M. hyopneumoniaeproteins. The experiment results showed that the present antigens wereable to induct immune response; wherein P46 vaccine and cocktail vaccine(contained the four antigens in one vaccine: P46, Tuf, MHP30, and NrdFC)showed the best induction effect and the induced immune response can bemaintained until 14-week age (FIG. 5).

3. Swine Challenge Tests.

Bayovac® MH-PRIT-5 vaccine prepared by using M. hyopneumoniae PRIT-5 wasused in this experiment as control for evaluating the immune efficacy ofthe present subunit vaccine and cocktail vaccine. 4-weeks SPF swine werebought from the second generation SPF swine house of Animal TechnologyInstitute Taiwan. All the swine were maintained and fed equally in theSPF swine house.

The M. hyopneumoniae bacterial broth used for the challenge tests wasprepared by: a swine lung tissue (about 3×3 cm²) infected by M.hyopneumoniae was collected and ground in 20 mL of Friis medium. Aftercentrifugation (148.8×g) for 10 minutes, the supernatant was transferredto a clean tube for centrifugation (7,870×g) for another 40 minutes.Then, the supernatant was discarded and the precipitation wasre-suspended with 6 mL of Friis medium to obtain a suspension. Afterthat, the suspension was filtered sequentially by 5 μm and 0.45 μmfilters to obtain the challenge bacterial broth needed for thisexperiment.

The vaccines (2 mL) were intramuscularly injected to the swine when theyreached 35-day age. Another injection was made at 49-day age via thesame way. At 63-day age, the immuned swine were anesthetized andchallenged by administering the challenge bacterial broth (5 mL) viatrachea. After 28 days from the day being challenged, the swine werescarified and the lungs thereof were collected for evaluating the immuneeffect. The evaluation of the immune effect was made according to thefollowing criteria: any one of meddle upper lobes and upper lobes of anyside of the lung observed of pathological trait was scored as 10 points;any of meddle upper lobe and diaphragmatic lobes of any side of the lungobserved of pathological trait was scored as 5 points. The full scorewas 55 points. The observation records were shown in Table 4.

TABLE 4 Evaluation of lung lesion in the challenge tests Antigen(s)Point of lung lesion MHP30 27 P46 25 NrdFC 26 Tuf 23 MHP30 + NrdFC +P46 + Tuf 17 PRIT-5 vaccine (positive control) 23 No vaccine used(negative control) 30

According to the data shown in Table 4, it is clear that the subunitvaccines of the present invention were able to provide equivalent immuneeffects as conventional vaccine (Bayovac® MH-PRIT-5). Furthermore, thecocktail vaccine (contained the four antigens in one vaccine: P46, Tuf,MHP30, and NrdFC) exhibited better immune effect significantly than theconventional vaccine. To sum up, the present proteins indeed aresuitable to be applied as active ingredients of vaccines and thoseproteins exhibited synergistic effect while being used in combination ascocktail vaccine.

Those having ordinary skill in the art can readily understand anypossible modifications based on the disclosure of the present inventionis without apart from the spirit of the present invention. Therefore,the examples above shall not be used for limiting the present inventionbut intend to cover any possible modifications under the spirit andscope of the present invention according to the claims recitedhereinafter.

What is claimed is:
 1. A composition, comprising: an active ingredient,comprising P46; wherein said P46 comprises SEQ ID NO: 01; and animmune-effective amount of a pharmaceutically acceptable adjuvant. 2.The composition of claim 1, said active ingredient further comprises aprotein selected from a group consisting of MHP30, NrdFC, and acombination thereof; wherein said MHP30 comprises SEQ ID NO: 03; andsaid NrdFC comprises SEQ ID NO:
 04. 3. The composition of claim 2,wherein each active ingredient protein is independently present at aconcentration of 20 to 2000 μg/mL based on the total volume of saidcomposition.
 4. The composition of claim 1, wherein saidpharmaceutically acceptable adjuvant is a complete Freund's adjuvant, anincomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanionadjuvant, a peptide, an oil emulsion, or a combination thereof.
 5. Thecomposition of claim 1, further comprising a pharmaceutically acceptableadditive.
 6. The composition of claim 5, wherein said pharmaceuticallyacceptable additive is a solvent, a stabilizer, a diluent, apreservative, an antibacterial agent, an antifungal agent, an isotonicagent, an absorption delaying agent, or a combination thereof.